We propose to determine which parameters known to correlate with transformation in various model systems are applicable to human cancer and at which stage in malignant progression such markers are expressed. To do this, we will develop and characterize syngeneic human epithelial cell lines representing various stages in malignant progression. We will concentrate on carcinomas of the bladder which are frequently found in association with benign lesions. By careful dissection of surgical specimens, we will be able to obtain normal, benign and malignant specimens from the same patient. We will also establish fibroblastic cell lines from the same patients for future studies involving interactions between stromal and epithelial cells. To develop the lines, we will utilize techniques similar to ones that we previously used to establish a series of epithelial cell lines derived from various carcinomatous and nonmalignant human tissue. We will also develop a media that is more suitable for culturing epithelial cells by determining the optimal nutritional and hormonal requirements for growth of normal, atypical and malignant cells. All of the lines will be characterized for malignant markers defined for various transformation systems including morphology, growth in methocel and on monolayers, saturation density, invasion of the chorioallantoic membrane, saturation density, presence of large external transformation sensitive protein (LETSP), ultrastructure by transmission electron microscopy (TEM) and surface morphology using scanning electron microscopy (SEM). To determine which characteristics are retained as cells grow in culture and develop into established lines, we will assay those parameters which can be measured in the primary specimens (TEM, SEM, presence of LETS protein) and all of the parameters as a function of passage.